TRFLP: amplify 16S with fluorophore tag, cut with restriction enzyme, run in capillary electrophoresis. See much less diversity with pooled 800 drops and less with pooled 100 drops. But need control of entire population cultivated under the same conditions.

I don’t know whether they can retrieve bacteria from individual droplets.

Michiel Kleerebezen: Microbiota of the small intestine are little-known because so hard to sample. Use people with ileocolostomy to access the small intestine. Can sample repeatedly, examine effects of dietary changes. Also volunteers who swallow a catheter for sampling.

Uses a human intestinal tract chip (HIT chip) to identify bacteria. Ileostomy effluent comes from the terminal portion of the small intestine, but its bacteria resemble that of the proximal ileum (quite a ways upstream). Now doing a metagenome and metetranscriptome analysis. Compare to large intesting data.

Metatranscriptome: Simple CHO utilization, fermentation. PTS transport very active. Biotin synthesis very active. Who's doing what? PTS exclusively streptococci and E coli and relatives. Hypothesize that lactate and acetate are excreted and used by other bacteria. Biotin made by everyone.

Who's there? Not many lactobacilli (yogurt) but if feed them a probiotic (10^9) lactobacilli see a spike to 50% lactobacilli in effluent over a day. How much did the lactobacilli grow? He didn't say - maybe I can ask later.

Study in 8 volunteers, crossover design. Fast, then drink every 30 min for 6 hr. Sample mucosa at 6 hr to see how host gut cells have changed their gene expression in response to the presence of the bacteria. (Lactobacillus acidophilus, L. casei, L. rhamnosus, each at 2-5 x 10^10.)

Showed 500-1000 differentially expressed host genes. Different gene-category effects by different bacteria. BUT sample size is small (8) and there are no error bars. He wisely claims to only have a "hypothesis-generating model".

Curtis Huttenhower (from Human Microbiome Project (HMP) Responsible for 30 posters at this meeting!): Overview: 300 people, 15-18 body sites, all being sequenced in different ways at various centres. Big questions: Who's there? What are they doing?

What to do with your (you the researcher) metagenome (from whole-genome shotgun sequencing)? Meed massive computational methods, to convert pinpricks of data into a big picture. He's telling us about metabolic reconstruction. Use cleanup analysis to convert sequence reads into measures of gene abundance, and then to smooth these into estimates of what pathways are present (encoded in present genes). Remove pathways only done by organisms absent from the sample. (Uses analysis methods taken from linguistics analysis!) The whole analysis was validated by sequencing and analyzing synthetic communities.

Data from 741 HMP samples: Lots of variation in which bacteria are present in different individuals, but much less variation in which pathways are present. Zero SPECIES of bacteria area present in all samples. But 19 PATHWAYS are present in all samples. Pathway abundances are consistent across individuals but variable across environments (body sites).

Methods from ecology. 'Alpha diversity'

To what extent do the differences in variability between 'Who's there?' (very variable) and "What are they doing?" (not variable) result from the nature of what is being measured rather than from ecological properties?

Look at pathways 'essential for life' and baxic metabolism. These are (unsurprisingly) very stable. If you zoom in on the metabolic details, see more variation in how specific pathways are being implemented.

Vaginal microbiome vignette: Very few genera present, mainly a few reference strains (Lactobacilli, Gardnerella). This affects the metabolic reconstruction.